1. Field of the Invention
This invention relates to a novel DNA construct, segment or fragment containing a promoter obtained from yeast and to an application thereof in genetic engineering.
2. Prior Art
With widespread utilization of recombinant DNA techniques, it has now become possible to produce useful polypeptides using prokaryotes or eukaryotes. Escherichia coli has thus far been employed in the large scale production of polypeptides. However, the use of eukaryotes is desired for the production of polypeptides particularly important in pharmacology. Yeasts, a group of eukaryotic microorganisms, have a number of similarities with mammalian cells and, therefore, are advantageous for use in the expression of genes coding for mammalian proteins. Further, yeasts do not contain endotoxin in their cells. Yeasts are easily cultivated. Culture of yeasts has been made from the past on a large, industrial scale, and its safety is confirmed. Additionally, a number of studies have been made to clarify their genetic biological mechanism. All the above circumstances surrounding yeasts have led to the utilization thereof as host organisms in genetic engineering.
Several yeast vectors for gene cloning are known at present. There are, however, few known yeast promoters capable of effectively expressing foreign genes.
Two acid phosphatases and two alkaline phosphatases are known to exist in a lysate of a strain of yeast Saccharomyces cerevisiae. The acid phosphatases are found on the surface of cells. The production of one of the acid phosphatases is suppressed by an inorganic phosphate, while the other acid phosphatase is constitutively produced. One of the alkaline phosphatases is a repressible one whose production is repressed by an inorganic phosphate and which has a wide substrate specificity. The other alkaline phosphatase is a specific p-nitrophenylphosphatase whose substrate is only p-nitrophenylphosphate, and which is constitutively produced. The mutants, pho5, pho4, pho2 and pho81 which lack repressible acid phosphatase activity have been isolated. The PHO5 gene is a structural gene for the repressible acid phosphatase, whereas the PHO4, PHO2 and PHO81 genes are genes which produce proteins regulating the expression of the repressible acid phosphatase structural gene PHO5. Further, the PHO4 and PHO81 genes serve to regulate the expression of repressive alkaline phosphatase structural gene similar to PHO5.
Various yeast vectors for gene cloning are known and may be utilized at present. In order to express native or endogeneous genes and foreign or exogeneous genes effectively, it is necessary to select a potent yeast promoter suitable for the host organisms to be used.